Rapid Sample Preparation Workflow for Serum Sample Analysis with Different Mass Spectrometry Acquisition Strategies

Serum proteomic analysis is essential for biomarker discovery and clinical diagnostics. Prior research has shown that traditional workflows require lengthy sample preparation, often involving overnight incubations. These methods limit throughput and delay clinical insights. While deep serum proteome coverage is achievable, the time and complexity of preparation remain barriers. High-throughput workflows are needed to enable rapid analysis in clinical settings. No prior work had resolved how to maintain depth while reducing preparation time. This gap motivated the development of a faster alternative. The need for rapid yet comprehensive serum analysis is clear in oncology and cardiovascular research.

This study aimed to develop a rapid sample preparation (RSP) workflow for serum proteomics. The goal was to reduce preparation time from overnight to less than 1.5 hours. The authors sought to maintain protein and peptide yield comparable to conventional methods. They also aimed to enable fast scanning of the serum proteome. The workflow was tested on HeLa cell lysates and serum samples. The RSP method was evaluated for its ability to support diverse mass spectrometry strategies. The study focused on reproducibility and depth of protein identification. The purpose was to meet clinical and research demands for speed and accuracy.

The RSP workflow was designed to replace overnight incubation with a streamlined process. Sample preparation was completed in under 1.5 hours using an ordinary system. The method was tested on HeLa cell lysates and serum samples. Protein and peptide yields were compared to conventional methods. LC-MS/MS analysis was used to assess the speed of serum proteome scanning. MRM was applied to quantify candidate biomarker peptides for cardiovascular disease. DIA acquisition mode was used to analyze depleted serum samples. The workflow was evaluated for reproducibility and depth of protein identification.

The RSP workflow produced comparable protein and peptide yields to conventional methods. A full serum proteome scan was completed in under 2 hours with 30 minutes of LC-MS/MS. Over 390 proteins were identified across eight orders of magnitude in abundance. More than 90 cancer-associated proteins and 50 FDA-approved biomarkers were detected. MRM quantification of eight CVD peptides showed high accuracy (CV% <10). After removing highly abundant proteins, over 1400 proteins were identified. DIA analysis of depleted serum quantified over 700 proteins. Differentially expressed proteins distinguished healthy and pancreatic cancer serum samples.

The RSP workflow is rapid and simple, meeting demands for both deep and fast serum proteome analysis. It reduces preparation time from overnight to less than 1.5 hours. The workflow maintains protein and peptide yields comparable to traditional methods. It supports diverse mass spectrometry acquisition strategies. The RSP method enables fast scanning and deep proteome coverage. It helps distinguish healthy and diseased serum samples with high reproducibility. The authors propose that it will be widely used in serum protein studies. They suggest it could accelerate biomarker discovery to clinical application.